CN101677989B - A drug composition for treatment and prevention of ischemic stroke and its preparation methods - Google Patents

Abstract

A drug composition for treatment and prevention of ischemic stroke and its preparation methods. The drug composition consists of Sedanolide 19-72%, Ligustilide 17-67%, Gastrodin 2-29% and solvent. It has a high purity of more than 98%. The drug composition can be prepared into different forms of drugs, including injection, and it can effectively alleviate symptoms of the disease.

Description

A kind of pharmaceutical composition of preventing and treating cerebral infarction and preparation method thereof

Technical field

The invention belongs to a kind of Chinese medicine monomer compositions, relate in particular to a kind of pharmaceutical composition of preventing and treating cerebral infarction and preparation method thereof.

Background technology

Apoplexy be in, the old people causes death and one of major cause of morbidity.Nearly 80% cerebral infarction case is because due to cerebral embolism or the atherosis thrombosis.Can cerebral ischemia be divided into the transient ischemic attack (TIA) that symptom was alleviated fully in 24 hours according to clinical symptoms and persistent period, reversibility ischemic neurological deficit (RIND) and the symptom duration property apoplexy just alleviated when symptom duration reached for 6 weeks.According to the data that WHO announces, in 57 countries, there are 40 countries to list the mortality rate of cerebrovascular the front three of the cause of death in, then account for the first place in Japanese and China.But existing medical treatment effect is bad, therefore is badly in need of the clinical application of exploitation better efficacy.

Medicinal Rhizoma Chuanxiong is Umbelliferae herbaceos perennial Rhizoma Chuanxiong (Ligusticum chuanxiong Hort) rhizome (English Rhizoma chuanxiong by name).The main chemical compositions of Rhizoma Chuanxiong is volatile oil, alkaloid, phenolic constituent, lactone, ferulic acid etc.The chemical constituent that wherein gets in the body and improve the Rhizoma Chuanxiong effective site of cerebral ischemia is the cnidium lactone compounds.Lactone wherein is sedanolide (4,5 dihydros-3-butyl benzene peptide, 4,5-dihydro-3-butyl-phthalide) (general formula (I) and ligustilide (4,5 dihydros-3-fourth is pitched basic benzene peptide, 4,5-dihydro-3-butylidene-phthal ide) (general formula (II).

Medicinal Rhizoma Gastrodiae (Gastrodia elata Blume; English by name Rhizoma Gastrodiae) be fungivorous plant such as perennial the orchid family height, Rhizoma Gastrodiae includes gastrodine, vanillin, succinic acid, cupreol, vitamin A appearance material, glycoside, alkaloid, phlegmatic temperament and gastrodia elata polysaccharide etc.Wherein gastrodine and gastrodia elata polysaccharide are its main components.At present can artificial synthetic gastrodine (gastrodin) (Zhou Shushun etc., Chinese Journal of Pharmaceuticals, 1985 05 phases)

Gastrodine (English name is Gastrodin), its chemical name is 4-hydroxy benzenes-β-D pyranglucoside.Molecular formula is: C13H1807.Rhizoma Gastrodiae has many-sided pharmacological action.Can recover the dysequilibrium between cerebral cortex excitement and process of inhibition, have calmness, sleep peacefully and central inhibitory action such as analgesia.The acute toxicity of gastrodine (Gastrodine) is very little.

China just is useful on the prescription of blood circulation promoting and blood stasis dispelling, endogenous wind stopping analgesic Rhizoma Chuanxiong and Rhizoma Gastrodiae, for example big Rhizoma Chuanxiong ball etc. from ancient times.In addition, also have other Chinese medicinal formulae of forming by Rhizoma Chuanxiong and Rhizoma Gastrodiae, for example " TIANXIONG injection ", this medicine belongs to Chinese medicine compound preparation, is made up of Rhizoma Chuanxiong, Rhizoma Gastrodiae two flavor Chinese medicines.The monitoring index of its Rhizoma Chuanxiong be ferulic acid (Huang Dongping etc. Chinese herbal medicine, 2004,35 (4), 409-411)." TIANXIONG Toutongning capsule for headache " is to be monarch drug with the Rhizoma Gastrodiae, by tens prescriptions formed of flavor Chinese medicines (Shen Yuhua etc. new Chinese medicine and clinical pharmacology, 1995,6 (3), 33-35)." TIANXIONG soft capsule " Main Ingredients and Appearance is Rhizoma Gastrodiae and Radix Et Rhizoma Rhei, wherein Rhizoma Gastrodiae and the Radix Et Rhizoma Rhei ratio in prescription be 3: 1 (Guo Zengjun etc. Chinese patent medicine 2005,27 (4), 467-468).These prescriptions are many to be main with the treatment migraine, and with the effective ingredient is not index in the preparation process.

Except that above-mentioned Chinese medicine compound preparation, it is the medicine of effective ingredient with Rhizoma Chuanxiong and Rhizoma Gastrodiae that the modern times also have some.(application number: 200610020605.2) disclosing with Rhizoma Gastrodiae and Rhizoma Chuanxiong is the pharmaceutical composition that active ingredient is formed in for example patent application " pharmaceutical composition and the preparation that comprise Rhizoma Gastrodiae and effective Chuanxiong component "; Patent application " big Rhizoma Chuanxiong dry powder formulations and method for preparing and application " (application number: 200610046747.6) disclose the dry powder formulations that is mixed and made into by Rhizoma Gastrodiae extract and Rhizoma Chuanxiong extract.Patent application " cnidium lactone of treatment ischemic heart desease and preparation method thereof " (application number: 03146029.1) disclose cnidium lactone, comprised two kinds of lactone compositions of sedanolide and ligustilide.Patent application " a kind of pharmaceutical composition of treating cerebrovascular disease and preparation method thereof " (application number: 200510101971.6) disclose compositions that ligustilide and senkyunolide form and " a kind of extract of cnidium lactone " (application number: 200610130680.4) disclose and relate to the application of cnidium lactone in the treatment ischemic cardiovascular.Patent " a kind of Chuanxiong rhizome volatile oil soft capsule and preparation method thereof " (application number: 200410037185.X) disclose a kind of method that adopts the carbon dioxide supercritical fluid extraction method to prepare Rhizoma Chuanxiong volatile oil.

There is following shortcoming in above-mentioned prior art: (1) drug ingredient purity is not high, can not process the Chinese medicine preparation of specific chemical components, particularly can't process the very high injection of specific chemical components and purity; (2) existing be to be monitoring index all in the Chinese medicine preparation of main control apoplexy with the Rhizoma Chuanxiong extract with the stable inadequately ligustilide of chemical property, the accuracy of influence detection.

Through retrieval, the Pharmaceutical composition that does not have discovery to form by sedanolide, ligustilide and gastrodine.

Summary of the invention

One of the object of the invention provides a kind of Pharmaceutical composition of preventing and treating cerebral infarction.

Second purpose of the present invention provides the method for preparing aforementioned pharmaceutical compositions.

Another object of the present invention provides the application of aforementioned pharmaceutical compositions in preparation treatment ischemic cerebral apoplexy Chinese medicine.

For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:

A kind of pharmaceutical composition of preventing and treating cerebral infarction, its constituent and percentage by weight thereof are:

Sedanolide 19～72%

Ligustilide 17～67%

Gastrodine 2～29%

The solvent surplus

Solvent described in the aforementioned pharmaceutical compositions is meant sodium carboxymethyl cellulose, tween 80 or plant wet goods.

Described vegetable oil is meant one or more in olive oil, Oleum Arachidis hypogaeae semen, Semen Maydis oil, soybean oil, Oleum Helianthi, Oleum Sesami, Oleum Ricini, almond oil or the Semen Persicae wet goods vegetable oil.

According to usual way, can be a kind of pharmaceutically acceptable dosage form with the dosage form of above-mentioned preparation of pharmaceutical compositions, as being injection, spray, capsule, drop pill, tablet, granule, Emulsion, dispersant, slow releasing agent etc.

The application of aforementioned pharmaceutical compositions in preparation treatment ischemic cerebral apoplexy Chinese medicine.

Sedanolide, ligustilide and gastrodine are pure article chemical compound in the aforementioned pharmaceutical compositions, and purity is all more than 98%, and gastrodine can be bought from market.

The preparation of drug combination method of above-mentioned control cerebral infarction is proportionally got sedanolide, ligustilide, gastrodine and cosolvent, and mix homogeneously gets final product then.

The method for preparing of above-mentioned sedanolide comprises the steps:

(1) exsiccant Rhizoma Chuanxiong root piece is crushed to the granule of diameter 1～3mm, places extraction kettle then;

(2) the temperature knob with extraction kettle is set to 42～58 ℃, heating; When the extraction kettle temperature is 42～58 ℃, open CO ₂Air intake pump;

(3) extraction and separation, CO ₂Air-flow gets into separation reactor I behind extraction kettle, get into separation reactor I I again; Arriving extraction kettle pressure is 18～32Mpa; Wherein separation reactor I pressure is 8～14Mpa, and temperature is 45～55 ℃, and the extraction time is 60～120min, CO ₂Flow is 220～350kg/h; Separation reactor I I pressure is 4～8Mpa, and temperature is 35～45 ℃, and the extraction time is 60～120min;

(4) in extraction kettle, feed intake once more after extraction for the first time and separating the is accomplished Rhizoma Chuanxiong root piece granule of same particle diameter, extraction second time and separation are carried out in repeating step (2)～(3);

(5) after extraction for the second time and the separation, respectively from the disposable entire contents of emitting of the discharging opening of separation reactor I and separation reactor I I;

(6) place separatory funnel until layering respectively above-mentioned two kinds of contents, collect supernatant liquid after the layering, the I level separation product (coming from separation reactor I) that obtains cnidium lactone respectively separates product (coming from separation reactor I I) with the II level;

(7) the I level of drawing the cnidium lactone of step (6) is separated the II level separation product of product or cnidium lactone or mixture 3～7ml adding petroleum ether of these two kinds of products: mixed solution to the 8～12ml of ethyl acetate (95: 5) shakes up; Sample introduction to Biotage 40+M positive liquid chromatograph silicagel column; Its mobile phase is the A petroleum ether, the B ethyl acetate; Flow velocity 50ml/min; Detect wavelength 280nm; Gradient elution, 0.01～12minA: B=95: 5,15～30minA: B=85: 15; Between 18～25min, collect eluent;

(8) repeating step (7) finishes I level and/or II level separation product sample introduction successively, gets eluent;

(9) the eluent rotary evaporation in 30～40 ℃ of water-baths with step (8) promptly gets the elementary purified product of sedanolide except that desolvating;

Elementary purified product 3～7ml adding dissolve with methanol to ShimadzuLC-8A anti-phase of (10) drawing in the step (9) prepares the liquid phase systems sample introduction; Wherein chromatographic column is Phenomenex Luna 10 μ m, 250mm * 50mm I.D.; Mobile phase is water (A) and methanol (B), and flow velocity is 80ml/min, isocratic elution, A: B=40: 60; The detection wavelength is 280nm; Between 18～26min, collect eluent;

(11) step (10) gained eluent rotary evaporation in 30～40 ℃ of water-baths is removed methanol; With residue with ethyl acetate extraction 3～5 times, extract;

(12) above-mentioned extract rotary evaporation in 30～40 ℃ of water-baths is got sedanolide except that desolvating.

The method for preparing of described ligustilide comprises the steps:

Step (1)～(6) are with sedanolide method for preparing step (1)～(6);

(7), collect the eluent between 6～12min with sedanolide step (7);

(8) rotary evaporation is except that desolvating in 30～40 ℃ of water-baths, and products obtained therefrom is ligustilide.

Supercritical carbon dioxide extraction still described in the above-mentioned method for preparing step (1) can be HA221-40-48 or HA421-40-963.13-3.43.

Described gastrodine can directly be bought from market, like the gastrodine or the acegastrodine of Kunming Medicine Group Stock Co., Ltd's production.

With the effective ingredient sedanolide and the ligustilide of the inventive method extraction, purification, purity is all more than 98%.Commercially available gastrodine (or acegastrodine) purity is more than 98%.

Advantage of the present invention and beneficial effect: (1) drug effect is high, can effectively alleviate the pathological change and the behavior disorder of cerebral infarction, and drug effect obviously is better than existing clinical application; (2) pharmaceutical composition drug effect of the present invention is definite, and consumption is little, and effect is strong; (3) purity is high, and each component of pharmaceutical composition of the present invention is pure article, and purity can be processed the preparation that comprises injection all more than 98%; (4) good stability, technology of the present invention reach preparation standard article level; (5) the present invention points out that the chemical property of sedanolide is more stable than ligustilide, is more suitable for the monitoring index as the Rhizoma Chuanxiong oil product quality.

Description of drawings

(wherein QHNP-1 is a ligustilide to Fig. 1 for the preparation spectrogram (forward HPLC method) from isolating ligustilide of supercritical extraction liquid and sedanolide product; QHNP-2 is a sedanolide)

Fig. 2 is the detection spectrogram (reverse hplc detection) of ligustilide

Fig. 3 is the preparation spectrogram (reverse hplc method) of the elementary purified product of sedanolide

Fig. 4 is the detection spectrogram (reverse hplc detection) of the final purified product of sedanolide

Fig. 5 is the infrared identification spectrogram of sedanolide

Fig. 6 is the infrared identification spectrogram of ligustilide

Fig. 7 is that the ultraviolet of sedanolide is identified spectrogram

Fig. 8 is that the ultraviolet of ligustilide is identified spectrogram

Fig. 9 is that the mass spectrum of sedanolide is identified spectrogram

Figure 10 is that the mass spectrum of ligustilide is identified spectrogram

Figure 11 is that the nuclear magnetic resonance, NMR of sedanolide is identified spectrogram (hydrogen spectrum)

Figure 12 is that the nuclear magnetic resonance, NMR of ligustilide is identified spectrogram (hydrogen spectrum)

Figure 13 is that the nuclear magnetic resonance, NMR of sedanolide is identified spectrogram (carbon spectrum)

Figure 14 is that the nuclear magnetic resonance, NMR of ligustilide is identified spectrogram (carbon spectrum)

The specific embodiment

Embodiment 1

The preparation of sedanolide

(1) the dry root piece 30kg that takes by weighing Rhizoma Chuanxiong is crushed to the granule of diameter 2.5mm, places the HA221-40-48 extraction kettle;

(2) rotation extraction kettle temperature setting knob to 50 ℃ when temperature reaches 50 ℃, is opened CO ₂Air intake pump;

(3) starting device carries out the extraction first time and separation; CO ₂Air-flow gets into separation reactor I, gets into separation reactor I I again behind extraction kettle; Extraction kettle pressure 25Mpa, separation reactor I pressure 11Mpa, 50 ℃ of separation reactor I temperature, extraction time 90min, CO ₂Flow 250kg/h, separation reactor I I pressure 6Mpa, 40 ℃ of separation reactor I I temperature;

(4) 30kg that feeds intake again, (1) set by step～(3) are carried out the extraction second time and are separated;

(5) respectively from the disposable entire contents of emitting of the discharging opening of separation reactor I and separation reactor I I;

(6) place separatory funnel until layering respectively above-mentioned two kinds of contents.Collect supernatant liquid after the layering, the I level separation product (coming from separation reactor I) that obtains cnidium lactone respectively separates product (coming from separation reactor I I) with the II level.

(7) the cnidium lactone II level of drawing in the step (6) is separated product 5ml, and the mixed solution (petroleum ether: ethyl acetate is 95: 5) that adds petroleum ether and ethyl acetate to 10ml, shakes up; Sample introduction to Biotage 40+M positive liquid chromatograph silicagel column; Its mobile phase is the A petroleum ether, the B ethyl acetate; Flow velocity 50ml/min; Detect wavelength 280nm; Gradient elution, 0.01～12minA: B=95: 5,15～30minA: B=85: 15; Between 19.6～23.2min, collect eluent (seeing Fig. 1 QHNP-2);

(8) repeating step (7) separates the product sample introduction with cnidium lactone II level and finishes, and gets eluent;

(9) step (8) is collected eluent rotary evaporation in 35 ℃ of water-baths promptly obtains the elementary purified product of sedanolide except that after desolvating;

(10) from the product of step (9), draw 5ml adding dissolve with methanol to Shimadzu LC-8A anti-phase and prepare the liquid phase systems sample introduction; Wherein chromatographic column is Phenomenex Luna 10 μ m, 250mm * 50mmI.D., and mobile phase is A water B methanol, flow velocity is 80ml/min, isocratic elution, A: B=40: 60, the detection wavelength is 280nm; Between 20.3～24.5min, collect the eluent (see figure 3);

(11) remove methanol at step (10) gained eluent rotary evaporation in 35 ℃ of water-baths, with residue with ethyl acetate extraction 3 times, extract;

(12) step (11) gained extract rotary evaporation in 35 ℃ of water-baths is obtained final products 347.3g except that desolvating.

(13) according to " the described detection method of Chinese pharmacopoeia version in 2005 appendix VI D " HPLC " detects resulting final products, and obtaining its purity is 99.17% (see figure 4).Learn that through infrared, ultraviolet, mass spectrum and nuclear magnetic resonance, NMR (comprising hydrogen spectrum and two kinds of methods of carbon spectrum) four big spectroscopy authentications this product is sedanolide (seeing Fig. 5, Fig. 7, Fig. 9, Figure 11 and Figure 13).

Embodiment 2

The preparation of sedanolide

(1) the dry root piece 30kg that takes by weighing Rhizoma Chuanxiong is crushed to the granule of diameter 1mm, places the HA221-40-48 extraction kettle;

(2) rotation extraction kettle temperature setting knob to 58 ℃ when temperature reaches 58 ℃, is opened CO ₂Air intake pump;

(3) starting device carries out the extraction first time and separates CO ₂Air-flow gets into separation reactor I, gets into separation reactor I I again behind extraction kettle, extraction kettle pressure 32Mpa, separation reactor I pressure 14Mpa, 55 ℃ of separation reactor I temperature, extraction time 120min, CO ₂Flow 280kg/h; Separation reactor I I pressure 8Mpa, 45 ℃ of separation reactor I I temperature;

(4) feed intake Rhizoma Chuanxiong granule 30kg again in the HA221-40-48 extraction kettle, repeating step (2)～(3) are carried out the extraction second time and are separated;

(5) respectively from the disposable entire contents of emitting of the discharging opening of separation reactor I and separation reactor I I;

(6) place separatory funnel until layering respectively above-mentioned two kinds of contents; Collect supernatant liquid after the layering, the I level separation product (coming from separation reactor I) that obtains cnidium lactone respectively separates product (coming from separation reactor I I) with the II level;

(7) above-mentioned two kinds of cnidium lactone products are fully mixed;

(8) mixed solution (petroleum ether: ethyl acetate is 95: 5) of absorption 5ml adding petroleum ether and ethyl acetate to 10ml, shakes up from the product of step (7); Sample introduction to Biotage 40+M positive liquid chromatograph silicagel column; Its mobile phase is petroleum ether (A) and ethyl acetate (B); Flow velocity 50ml/min; Detect wavelength 280nm; Gradient elution is from 0.01～12minA: B=95: 5,15～30minA: B=85: 15; Between 18～25min, collect eluent;

(9) collected eluent rotary evaporation in 30 ℃ of water-baths is promptly obtained the elementary purified product of sedanolide except that after desolvating;

(10) from the product of step (9), draw 5ml adding dissolve with methanol to Shimadzu LC-8A anti-phase and prepare the liquid phase systems sample introduction; Wherein chromatographic column is Phenomenex Luna 10 μ m, 250mm * 50mmI.D., and mobile phase is A water B methanol, flow velocity is 80ml/min, isocratic elution, A: B=40: 60, the detection wavelength is 280nm; Between 18～26min, collect eluent;

Rotary evaporation is removed methanol in (11) 30 ℃ of water-baths, then with residue with ethyl acetate extraction 5 times, extract;

(12) extract rotary evaporation in 30 ℃ of water-baths is got sedanolide 309g except that desolvating.

Embodiment 3

The preparation of ligustilide comprises the steps:

(1) takes by weighing exsiccant Rhizoma Chuanxiong root piece 30kg, be crushed to the granule of diameter 3mm, place the HA221-40-48 extraction kettle;

(2) the extraction kettle temperature is 42 ℃, when temperature reaches setting value, opens CO ₂Air intake pump;

(3) extraction kettle pressure 18Mpa, separation reactor I pressure 8Mpa, 45 ℃ of separation reactor I temperature, extraction time 60min, CO2 flow 220kg/h, separation reactor I I pressure 5Mpa, 40 ℃ of separation reactor I I temperature are carried out the extraction first time and separation;

Step (4)～(7) are with step (4)～(7) of embodiment 2;

(8) from the product of step (7), draw the 5ml sample introduction to Biotage 40+M positive liquid chromatograph silicagel column; Chromatographic condition is with embodiment 2; Between 7～10.6min, collect eluent, obtain final products 498.2g (seeing the QHNP-1 among Fig. 1).

(9) according to " the described detection method of Chinese pharmacopoeia version in 2005 appendix VI D " HPLC " detects resulting final products, and obtaining its purity is 98.32%%.Learn that through infrared, ultraviolet, mass spectrum and nuclear magnetic resonance, NMR (comprising hydrogen spectrum and two kinds of methods of carbon spectrum) four big spectroscopy authentications this product is ligustilide (seeing Fig. 6, Fig. 8, Figure 10, Figure 12 and Figure 14).

Embodiment 4

Get (back with) sedanolide (proportion 0.95, back with) 4.2g, ligustilide 1.0g (proportion 0.95, back with) and the gastrodine 0.6g of embodiment 1～3 preparation respectively, join in the 20g Pure Olive Oil, ultrasonic mix homogeneously, present composition A 25.8g.

Embodiment 5

Get sedanolide 0.6g, ligustilide 1.8g and gastrodine 0.75g respectively, join in the 20g soybean oil, ultrasonic mix homogeneously gets present composition B 23.15g.

Embodiment 6

Get sedanolide 3.6g, ligustilide 3.3g and gastrodine 2.9g respectively, add pure Oleum Arachidis hypogaeae semen to 20ml, ultrasonic mix homogeneously gets present composition C 29.8g.

Embodiment 7

Take by weighing sedanolide 2.8g respectively, ligustilide 1.2g, gastrodine 0.08g grinds well, and adds 0.5% sodium carboxymethyl cellulose to 21.6ml, gets present composition D 25.68g.

Embodiment 8

Get sedanolide 1.4g, ligustilide 3.0g and gastrodine 0.1g respectively, join 2% sodium carboxymethyl cellulose to 20ml, ultrasonic mix homogeneously gets present composition E 25.5g.

Embodiment 9

The sedanolide stability experiment

According to high effective liquid chromatography for measuring (seeing an appendix VI of Chinese Pharmacopoeia version in 2005 D).

Chromatographic condition and system suitability test: select Tianjin, island LC-2010 performance liquid chromatographic column for use.With the octadecylsilane chemically bonded silica is filler (Diamonsil C18,4.6 * 150mm, 5 μ m); With acetonitrile-water (42: 58) is mobile phase; 35 ℃ of column temperatures; Detect wavelength 280nm, 230nm; Flow velocity 1ml/min.Number of theoretical plate calculates by the sedanolide peak and is not less than 16000.

The preparation of standard solution: get the prepared sedanolide of embodiment 1, accurately claim surely, add methanol and process the solution that every 1ml contains sedanolide 0.08mg respectively, promptly get purity greater than 99% standard solution.

The preparation of need testing solution: it is an amount of to get the sedanolide that embodiment 1 obtains, accurate claim fixed, with dissolve with methanol and quantitatively dilution process the solution that contains 0.08mg among every 1ml, as the sedanolide need testing solution.

Assay method: accurate sedanolide standard substance and each 10ul of need testing solution of drawing, inject chromatograph of liquid, get final product according to the said method mensuration of embodiment 1 step (13).

Table 1 sedanolide stability experiment result (normalization purity testing %, 280nm)

Condition of storage	0 day (degree of grade)	5 days (degree of grade)	10 days (degree of grade)	20 days (gradient)	30 days (gradient)
						The room temperature lucifuge is not filled nitrogen	99.028	98.288	98.228	98.270	98.286
The room temperature lucifuge is filled nitrogen	99.028	98.947	98.909	99.050	99.124
						Light at room temperature is according to not filling nitrogen	99.028	98.210	98.070	-	98.142
Light at room temperature is according to filling nitrogen	99.028	98.825	98.824	-	99.059
						40 ℃ of lucifuges are filled nitrogen	99.028	98.856	98.966	-	99.024
60 ℃ of lucifuges are filled nitrogen	99.028	98.717	98.896	-	98.965
						The cold preservation lucifuge is filled nitrogen	99.028	98.983	99.038	99.022	99.110

The stability of result's (seeing table 1) sedanolide of the present invention mainly receives the influence of high temperature, illumination and oxygen.No matter in 30 days, fill under the condition of storage of nitrogen lucifuge, be room temperature, cold preservation or freezing, and the stability of sedanolide is not had influence (relative standard deviation of content＜2%) basically.Explain that sedanolide character is more stable, keep in Dark Place that purity can not descend in 30 days, can be used as standard substance and use as long as fill nitrogen.

Embodiment 10

The ligustilide stability experiment

According to " the high effective liquid chromatography for measuring shown in an appendix VI of Chinese pharmacopoeia version in 2005 D.

Chromatographic condition and system suitability test: select Tianjin, island LC-2010 performance liquid chromatographic column for use.With the octadecylsilane chemically bonded silica is filler (Diamonsil C18,4.6 * 150mm, 5 μ m); With acetonitrile-water (42: 58) is mobile phase; 35 ℃ of column temperatures; Detect wavelength 280nm, 230nm; Flow velocity 1ml/min.

The preparation of standard solution: get the prepared ligustilide of embodiment 3, accurately claim surely, add methanol and process the solution that every 1ml contains ligustilide 0.4mg, promptly get purity greater than 99% standard solution.

The preparation of need testing solution: it is an amount of to get embodiment 3 gained ligustilides, and accurate the title decides, and processes the solution that contains 0.4mg among every 1ml with dissolve with methanol and quantitative dilution, as the ligustilide need testing solution.

Assay method: draw standard substance and each 10ul of need testing solution, inject chromatograph of liquid and measure according to the said method of embodiment 3 steps (9).

Result's (seeing table 2).Ligustilide stability of the present invention mainly receives illumination, temperature and oxidation affects; The room temperature condition held after 10 days purity drop to below 95% from 98%.Show that the ligustilide standard substance must fill storage under nitrogen, lucifuge, cold preservation or the freezing conditions.Must fresh.Each preparation amount is no more than 5 days use amounts.

Table 2 ligustilide stability experiment result (normalization purity testing %, 280nm)

Condition of storage	0 day (degree of grade)	5 days (degree of grade)	10 days (degree of grade)	20 days (gradient)	30 days (gradient)
						The room temperature lucifuge is not filled nitrogen	98.264	95.707	97.214	95.303	94.552
The room temperature lucifuge is filled nitrogen	98.264	97.892	98.345	96.546	95.908
						Light at room temperature is according to not filling nitrogen	98.264	95.534	94.112	-	27.669
Light at room temperature is according to filling nitrogen	98.264	96.978	95.202	-	18.084
						40 ℃ of lucifuges are filled nitrogen	98.264	97.999	97.971	-	90.114
60 ℃ of lucifuges are filled nitrogen	98.264	96.948	96.836	-	60.214
						The cold preservation lucifuge is filled nitrogen	98.264	97.304	98.170	97.442	97.150
Freezing lucifuge is filled nitrogen	98.264	97.657	98.290	97.662	97.403

Embodiment 11

Pharmaceutical composition of the present invention is to the influence of line bolt method cerebral ischemic model

(1), test method is carried out according to following method:

(1). divide into groups and administration: select for use 150 body weight to make an experiment the healthy male SPF/VAF of 240～250g level Wistar rat.(Beijing Vital River Experimental Animals Technology Co., Ltd. provides, the quality certification number: SCXK capital 2002-0003).Be divided into 9 groups at random, i.e. blank group, model control group, MAILUONING positive controls 6ml/kg, compositions A, B, each two dosage (6mg/kg and 12/kg) group of C.The compound method of compositions A, B, C is seen embodiment 4～6

Performed the operation preceding 1 hour and 2 hours a kind of compositionss of lumbar injection or normal saline or positive drug after the perfusion again.Positive drug is chosen the Western medicine MAILUONING ZHUSHEYE commonly used of clinical treatment cerebral infarction, and (10ml/ props up, and Jinling Pharmaceutical Co., Ltd., Jinling Pharmaceutical Factory produces, lot number: 200503011).Blank group and model control group lumbar injection equal-volume normal saline.

(2). line bolt preparation: the diameter of selecting for use Japan to produce is that the nylon yarn of 0.23mm is moulded linear and is cut into the line segment of 5cm; Front end with fine sandpaper polishing, dip in glue and make it level and smooth; Marking subsequent usely apart from front end 1.6cm, 1.8cm, 2.0cm place, facing the time spent nylon yarn front end is dipped in taking heparin.

(3). model copy: 400mg/kg lumbar injection 10% chloral hydrate anesthesia; Cervical region medisection exposes left carotid (being called for short CCA), external carotid artery (being called for short ECA), internal carotid artery (being called for short ICA); Ligation CCA near-end, ECA root; Cut an angle at CCA ligation place far-end, insert nylon yarn, go into cranium to anterior cerebral artery (ACA) through ICA, the blood flow of blocking-up MCA through the CCA crotch; The average insertion depth of nylon yarn is 18.5 ± 0.5mm, ligation ICA.Skin suture, it is terminal to expose nylon yarn; Nylon yarn is extracted after 2 hours in ischemia, observed the variation of the different time-histories in perfusion back again; Sham operated rats is not inserted nylon yarn.

(2), observation index:

(1). neuroethology scoring: adopt Pyatyi point system (0-4 branch).In the whole behavioristics's observation of carrying out animal latter stage of perfusion again.Carry about 1 chi of Mus tail built on stilts, observe two forelimb situations; Rat is placed the level ground, promote its both shoulders, observing the both sides resistance has zero difference; Rat places ground, observes its walking situation.Mark is high more, shows that its neurobehavioral damage is serious more.(by best result)

A. behavior fully normally is 0 minute;

B. mention Mus tail built on stilts, operation offside forelimb inward turning, interior receipts person are 1 minute;

C. rat is to ground, and with its drag of hands extruding both sides inspection, operation offside drag descender is 2 minutes;

D. rat is observed its walking to ground, around the operation offside person of turn-taking, is 3 minutes;

E. damage is extremely serious, can't the autonomic activities person, and be 4 minutes.

(2). the cerebral infarct size test: broken end is got brain after 24 hours, and the left side brain is cut to thick totally 5 of 2mm, thick totally 1 of 4mm.Through 37 ℃ of lucifuge dyeing of 4% red tetrazolium (TTC) 30 minutes; Whenever stirred once therebetween at a distance from 5 minutes.After TTC dyeing, the normal structure engrain takes on a red color, and infarction tissue is white in color.With every group of brain sheet marshalling, the preservation of taking pictures.Application image analytical system software (SPOT 3.5 softwares) is handled and statistics.The infarct size and the final stack of calculating every are converted into infarct volume.Infarct volume is represented with shared Interhemispheric percentage rate.Computing formula is following:

Volume * 100% of cerebral infarct volume (%)=(volume-operation side hemisphere of operation offside hemisphere does not block the volume of part)/operation offside hemisphere

(3). brain water content is measured:

After pouring into 24 hours again, broken end is got brain at once, removes tractus olfactorius, XIAONAO and low brain stem, the side of separately performing the operation and normal side hemisphere, weigh respectively (weight in wet base).Place 95 ℃ of baking boxs to toast 24 hours to constant weight, take by weighing brain heavy (dry weight) once more.Brain water content calculates by following formula:

Brain water content (%)=(weight in wet base-dry weight)/weight in wet base * 100%

Experimental data is represented with X ± SD, through t check comparable group differences.

Table 3. Pharmaceutical composition of the present invention is to line bolt method cerebral ischemic model therapeutic test result

Group (N=10)	Dosage (mg (ml)/kg)	Brain per cent moisture (%)	Infarct size (% ±SD)	24 hours behavior scorings (0-4 score value)
					Blank group model matched group MAILUONING compositions A compositions A compositions B compositions B compositions C compositions C	6ml 12.0 6.0 12.0 6.0 12.0 6.0	78.46±0.19 79.28±0.30△△ 79.17±0.42 79.02±0.12* 79.15±0.20 79.75±0.53 79.18±0.43 79.20±0.34 79.29±0.23	2.90±1.98 34.04±9.65△△ 13.75±2.49** 17.32±13.44** 13.03±8.06** 11.75±9.91** 18.58±8.47** 13.31±1.16** 13.46±3.10**	0 1.90±0.88△△ 1.80±0.42 2.30±0.95 1.69±0.63 1.70±0.67 1.50±0.53 1.80±0.63 1.60±0.70

Annotate: the △ △ P that compares with the blank group＜0.01 △ △ P＜0.05;

* * P＜0.01*P＜0.05 of comparing with model control group;

Result's (seeing table 3) A of the present invention, B, three groups of Pharmaceutical compositions of C and positive drug group all can be improved intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size.Wherein best with heavy dose of (12mg/kg body weight) effect of compositions B, drug effect is better than clinical application MAILUONING ZHUSHEYE commonly used.Explain that Pharmaceutical composition of the present invention can be used for treating cerebral infarction, alleviates cerebral infarct size.

Embodiment 12

Pharmaceutical composition of the present invention is to the influence of line bolt method cerebral ischemic model

(1), test is carried out according to following method:

(1) divides into groups and 78 of the healthy male SD rats of administration: 200～220g, be divided into 6 groups at random, be respectively compositions A, B, the C group of normal control group, model group, positive control drug ligustrazine group and the embodiment of the invention 4～6.Positive control drug ligustrazine group is selected from Ligustrazine Phosphate pill, the 50mg/ sheet, and 100 slices/bottle, Beijing, Beijing pharmaceutical factory produces, lot number: 040603.Gastric infusion is 7 days in advance, once a day, and each dosage 50mg/kg.2h was administered once again before broken end was got brain.Normal control group and model group are irritated stomach equivalent normal saline.

(2) experimental technique: 4% chloral hydrate (350mg/kg, ip) anesthesia.The rat dorsal position is fixed,, separate right carotid and wear 2 sutures subsequent use, separate external carotid artery again, the proximal part ligation along neck medisection.Branch of internal carotid artery and next door thereof (pterygoid process arteria palatina) is isolated in the external carotid artery below, under branch, wears a suture, in the ligation of proximal part crotch.Use the suture ligation at isolating common carotid artery proximal part, distal end is used the bulldog clamp blocking blood flow, between this; Cut an osculum, an end is heated into round bead shape, and (nylon wire (4-0) of diameter＜0.3mm) inserts osculum, removes bulldog clamp; Nylon wire slowly is pushed into preceding cerebral arteries (about 20mm); Toward pulling back about 2mm promptly to the middle cerebral artery mouth, be about 17mm (the aortic bifurcation place is counted inside and outside neck) again, fixedly nylon wire is excellent with the suture ligation.Suture muscles and skin.In the operation process, visual field temperature keeps 37 ℃.Postoperative 24h carries out behavioristics's scoring, after the scoring, puts to death rat, and broken end is got brain.Matched group is plug wire not, and postoperative 24h carries out behavioristics scoring back and puts to death, and broken end is got brain.Below operation and computational methods are equal to relative section content among the embodiment 11.

(2), observation index: with embodiment 11 observation index (2)

Result's (seeing table 4) A of the present invention, B, three groups of Pharmaceutical composition oral agents of C and positive drug group prevention administration all can improve intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size in 7 days; A of the present invention, B, three groups of Pharmaceutical composition drug effects of C obviously are better than clinical application Ligustrazine Phosphate pill commonly used.Explain that Pharmaceutical composition of the present invention can prevent and treat cerebral infarction, alleviate cerebral infarct size.Drug effect obviously is better than clinical application Ligustrazine Phosphate pill (P＜0.01) commonly used.

Table 4 Pharmaceutical composition of the present invention is to line bolt method cerebral ischemic model result of the test

Group	Dosage (mg/kg)	Number of animals (only)	Cerebral infarct size % ( ±SD)
				The normal control group	/	10	0
Model group	50	10	27.67±0.05*
				Compositions A	50	10	21.73±0.04**
Compositions B	50	10	21.48±0.04**
				Compositions C	50	10	21.36±0.04**
Positive drug group (ligustrazine)	50	10	25.20±0.03*

Annotate: * P＜0.05 of comparing with model control group, * * P＜0.01 of comparing with model control group

Embodiment 13

Pharmaceutical composition of the present invention is to the influence of tri-chlorination iron processes cerebral ischemic model

(1), experiment is carried out as follows:

(1) grouping and administration: 110 body weight are divided into 9 groups the healthy male SD of 180～200g rat by body weight at random.Blank group, model control group, ligustrazine phosphate 120mg/ml group (medicine source is with embodiment 12), each composition component are 80mg/ml, two dose groups of 40mg/ml, 12 every group.Every day oral administration once, continuous 30 days, blank control group, model control group were taken the equal-volume edible oil.

(2) experimental technique: after 1 hour, the chloral hydrate solution of pressing 350mg/ml lumbar injection 12% is with rat anesthesia in the last administration, and lateral position is fixed, and under operating microscope, operates.The eye corner of the eyes and external auditory meatus line mid point are made a vertical incision that is about 1cm; Expose also and peel off temporalis, bore the aperture of the about 2mm of a diameter at 1mm place, oval foramen side front upper place with dental drill, puncture meninx with fine needle; Expose arteria cerebri media (MCA); The filter paper bar that dips in 50% liquor ferri trichloridi is spread on the arteria cerebri media, take out behind the 30min, this moment, arteria cerebri media became black or white.Sew up the incision.Broken end is got brain behind the 24h.Below operation and computational methods etc. are seen relative section content among the embodiment 11.

The neuroethology test is carried out behind operation 6h and 24h respectively.Standards of grading and method are with embodiment 11 observation index (1).

(2), observation index: with embodiment 11 observation index (1), (2)

Result's (seeing table 5) respectively organizes Pharmaceutical composition oral agents and positive drug group and prevents administration 30 days to compare all with model group continuously can obviously to improve intraluminal middle cerebral artery occlusion in rats focal cerebral ischemia infarct size.Wherein present composition A drug effect obviously is better than clinical application Ligustrazine Phosphate pill (P＜0.05) commonly used.Because the positive drug group of Isodose can't embody drug effect, have to escalated dose.And present composition group dosage 1/3～2/3 can manifest similar drug effect.Explain that Pharmaceutical composition oral agents of the present invention long term administration can prevent and treat cerebral infarction, alleviates cerebral infarct size.Drug effect is better than or is not less than uses the clinical application Ligustrazine Phosphate pill always.

Table 5. pharmaceutical composition of the present invention prevention administration 30 days is to the cerebral ischemic model result of the test

Annotate: △ △ P＜0.01 of comparing with the blank group

* P＜0.05 of comparing with model control group

Embodiment 14

The acute toxicity test in mice of pharmaceutical composition of the present invention

(1) experiment and computational methods:

Adopt SPF level Kunming kind white mice, disposable oral administration.With the trial test result is test dose, and the agent distance is 1: 0.90.

The medicinal liquid compound method is following: accurately take by weighing sedanolide 2.501g, ligustilide 2.502g, and gastrodine 0.7502g, add an amount of 2% sodium carboxymethyl cellulose hydrotropy behind the mixing, process the solution that concentration is 0.0575g/g after fully grinding well; Each organizes dosage respectively by the aqueous solution of dosage shown in the table 7 with 2% sodium carboxymethyl cellulose) dilution, every 20g body weight oral administration 0.8ml; Observed 48 hours continuously.

Animal is divided into 9 groups at random by the body weight of fasting after 16 hours, 20 every group, male and female half and half; Agent was apart from 1: 0.9 between group; Respectively the thing that tried of above-mentioned variable concentrations is irritated stomach by 0.8ml/20g, observe at once and tried the toxic reaction that each treated animal occurs behind the thing, observed continuously 48 hours.Toxic reaction and the death condition of record animal.Calculate the oral LD of mice with the Bilss method ₅₀Dosage and 95% fiducial limit.

Computing formula is following:

Regression coefficient: $b=\frac{6\mathrm{\Σ\; Yr}}{\mathrm{IK}(K^2-1)}$

Median lethal dose(LD 50): ${\mathrm{<figure-callout\; id="50"\; label="LD"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">LD</figure-callout>}}_{50}=\mathrm{Ant}\mathrm{Log}[\stackrel{\&OverBar;}{X}+\frac{1}{b}(5-\stackrel{\&OverBar;}{Y})]$

Log LD ₅₀Standard error: ${\mathrm{<figure-callout\; id="50"\; label="Sx"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">Sx</figure-callout>}}_{50}=\frac{1}{b}\&CenterDot;\sqrt{\frac{1}{\mathrm{N\&Sigma;\; w}}}$

LD ₅₀95% average fiducial limit: ${\mathrm{<figure-callout\; id="95"\; label="L"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">L</figure-callout>}}_{95}={\mathrm{<figure-callout\; id="50"\; label="LD"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">LD</figure-callout>}}_{50}\&PlusMinus;4.5{\mathrm{<figure-callout\; id="50"\; label="LD"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">LD</figure-callout>}}_{50}\&CenterDot;\frac{1}{b\sqrt{\mathrm{N\&Sigma;\; w}}}$

LD ₅₀99% average fiducial limit: $L_{99}={\mathrm{<figure-callout\; id="50"\; label="LD"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">LD</figure-callout>}}_{50}\&PlusMinus;5.9{\mathrm{<figure-callout\; id="50"\; label="LD"\; filenames="HPCT139291908150143000E000051.png,HPCT139291908150143000E000061.png"\; state="\{\{state\}\}">LD</figure-callout>}}_{50}\&CenterDot;\frac{1}{b\sqrt{\mathrm{N\&Sigma;\; w}}}$

Result's (seeing table 6) shows the oral LD of mice of pharmaceutical composition of the present invention ₅₀Value is 0.7276g/kg; LD ₅₀95% average credible 0.7276 ± 0.0470g/kg (scope: 0.6806-0.7746g/kg) that is limited to; LD ₅₀99% average credible 0.7276 ± 0.0616g/kg (scope: 0.6660-0.7892g/kg) that is limited to.Agent was apart from 1: 0.9 between group.

This test is for being the acute toxicity test of the pure article of chemical grade.The result shows that pharmaceutical composition safety of the present invention is good, for clinical medicine dose provides the valuable reference data.

The oral LD of table 6 mice ₅₀Dosage and 95% fiducial limit experimental result

No.	Dosage g/kg	Logarithm metering X	n	The dead animal number	Mortality rate P	The Y of probit	Weight coefficient W	R	RY
											1	1.000	0	20	20	1.00	7.620	?0.116	9-1＝8	60.960
2	0.900	-0.0458	20	19	0.95	6.645	?0.224	9-3＝6	39.870
										3	0.810	-0.0915	20	18	0.90	5.282	?0.342	9-5＝4	25.128
4	0.729	-0.1373	20	15	0.75	5.674	?0.538	9-7＝2	11.348
										5	0.656	-0.1831	20	12	0.60	5.253	?0.621	9-9＝0	0.000
6	0.590	-0.2291	20	7	0.35	4.603	?0.603	9-11＝-2	-9.206
										7	0.531	-0.2749	20	5	0.25	4.326	?0.538	9-13＝-4	-17.304
8	0.478	-0.3206	20	3	0.15	3.964	?0.426	9-15＝-6	-23.784
										9	0.430	-0.3665	20	0	0.00	2.380	?0.116	9-17＝-8	-19.04
		＝-0.1832				＝5.1941	?∑＝3.524		67.972

i＝log(1/0.9)＝0.0458

b＝12.3675

LD ₅₀＝0.6326g·kg ^-1

L ₉₅=0.6326 ± 0.0274gkg ^-1(scope: 0.6052-0.6600gkg ^-1)

L ₉₉=0.6326 ± 0.0359gkg ^-1(scope: 0.5967-0.6685gkg ^-1)

Sx ₅₀＝0.0096

Claims (10)

1. a pharmaceutical composition of preventing and treating cerebral infarction is characterized in that, contains: purity is 99.17%, proportion is 0.95 sedanolide 4.2g, and purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 1.0g and gastrodine 0.6g.

2. the method for preparing that a kind of pharmaceutical composition of preventing and treating cerebral infarction according to claim 1 proposes, it is characterized in that; Purity be 99.17%, proportion is 0.95 sedanolide 4.2g; Purity is 98.32%, proportion is that 0.95 Artemisia benzene lactone 1.0g and gastrodine 0.6g join in the 20g Pure Olive Oil; Ultrasonic mix homogeneously obtains pharmaceutical composition 25.8g.

3. a pharmaceutical composition of preventing and treating cerebral infarction is characterized in that, contains: purity is 99.17%, proportion is 0.95 sedanolide 0.6g, and purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 1.8g and gastrodine 0.75g.

4. the method for preparing that a kind of pharmaceutical composition of preventing and treating cerebral infarction according to claim 3 proposes; It is characterized in that; Purity be 99.17%, proportion is 0.95 sedanolide 0.6g, purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 1.8g and gastrodine 0.75g, joins in the 20g soybean oil; Ultrasonic mix homogeneously obtains pharmaceutical composition 23.15g.

5. a pharmaceutical composition of preventing and treating cerebral infarction is characterized in that, contains: purity is 99.17%, proportion is 0.95 sedanolide 3.6g, and purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 3.3g and gastrodine 2.9g.

6. the method for preparing that a kind of pharmaceutical composition of preventing and treating cerebral infarction according to claim 5 proposes, it is characterized in that; Purity be 99.17%, proportion is 0.95 sedanolide 3.6g; Purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 3.3g and gastrodine 2.9g; Join in the pure Oleum Arachidis hypogaeae semen of 20ml, ultrasonic mix homogeneously obtains pharmaceutical composition 29.8g.

7. a pharmaceutical composition of preventing and treating cerebral infarction is characterized in that, contains: purity is 99.17%, proportion is 0.95 sedanolide 2.8g, and purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 1.2g and gastrodine 0.08g.

8. the method for preparing that a kind of pharmaceutical composition of preventing and treating cerebral infarction according to claim 7 proposes, it is characterized in that; Purity be 99.17%, proportion is 0.95 sedanolide 2.8g; Purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 1.2g and gastrodine 0.08g; Mix homogeneously, adding concentration is 0.5% sodium carboxymethyl cellulose 21.6ml, obtains pharmaceutical composition 25.68g.

9. a pharmaceutical composition of preventing and treating cerebral infarction is characterized in that, contains: purity is 99.17%, proportion is 0.95 sedanolide 1.4g, and purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 3.0g and gastrodine 0.1g.

10. the method for preparing that a kind of pharmaceutical composition of preventing and treating cerebral infarction according to claim 9 proposes, it is characterized in that; Purity be 99.17%, proportion is 0.95 sedanolide 1.4g; Purity is 98.32%, proportion is 0.95 Artemisia benzene lactone 3.0g and gastrodine 0.1g; Mix homogeneously, adding concentration is 2% sodium carboxymethyl cellulose 20ml, obtains pharmaceutical composition 24.5g.

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